Methods for detecting dna mutations using mitra tip extraction

ABSTRACT

The present disclosure provides rapid and non-invasive methods for determining whether a patient exhibiting cancer symptoms, or at risk for hereditary cancers such as breast cancer, ovarian cancer, colon cancer, or skin cancer, will benefit from treatment with one or more therapeutic agents. These methods are based on detecting hereditary cancer-related mutations in small-volume dried biological fluid samples that are collected using a volumetric absorptive microsampling device (e.g., MITRA Tip). Kits for use in practicing the methods are also provided.

TECHNICAL FIELD

The present disclosure provides methods for determining whether apatient exhibiting cancer symptoms, or at risk for hereditary cancerssuch as breast cancer, ovarian cancer, colon cancer, or skin cancer,will benefit from treatment with one or more therapeutic agents. Thesemethods are based on detecting hereditary cancer-related mutations insmall-volume dried biological fluid samples that are collected using avolumetric absorptive microsampling device. Alterations in targetnucleic acid sequences corresponding to one or more genes associatedwith hereditary cancers may be detected using next generation sequencing(NGS). Kits for use in practicing the methods are also provided.

BACKGROUND

The following description of the background of the present disclosure isprovided simply to aid the reader in understanding the disclosure and isnot admitted to describe or constitute prior art to the presentdisclosure.

Hereditary cancers account for 5-10% of all cancers, and arise due tohighly penetrant germline mutations. The most common hereditary cancersare breast cancer, ovarian cancer, colon cancer, and skin cancer.Inheriting a genetic mutation or pathogenic variant increases apatient's risk for developing cancer. Accordingly, determining whether acancer arises due to an inherited pathogenic variant may be useful inassessing a patient's risk for developing cancer and may help uncoveroptions for cancer screening, prevention, and therapy.

Next generation sequencing (NGS) is extensively used in cancerdiagnostics because of its ability to detect multiple gene alterationsin a single assay in a high throughput fashion. However, the proceduresassociated with collecting and preparing nucleic acids from biologicalsamples (e.g., blood) are usually cumbersome, and often requirespecialized equipment or technical skill. Further, critically illpatients, such as cancer patients, are unable to provide large volumesof blood for recurrent testing.

Thus, there is a need for rapid and non-invasive methods for determiningwhether a patient is at risk for hereditary cancers such as breastcancer, ovarian cancer, colon cancer, or skin cancer.

SUMMARY

In one aspect, the present disclosure provides a method for detecting atleast one mutation in a plurality of hereditary cancer-related genes ina dried biological fluid sample comprising (a) extracting genomic DNAfrom a dried biological fluid sample eluted from an absorbent tip of amicrosampling device; (b) generating a library comprising ampliconscorresponding to each of the plurality of hereditary cancer-relatedgenes, said plurality of hereditary cancer-related genes comprising APC,ATM, BARD1, BMPR1A, BRCA1, BRCA2, BRIP1, CDH1, CDK4, CDKN2A (p14ARF andp16), CHEK2, EPCAM MEN1, MLH1, MSH2, MSH6, MUTYH, NBN, NF1, PALB2, PMS2,POLD1, POLE, PTEN, RAD51C, RAD51D, RET, SDHB, SDHC, SDHD, SMAD4, STK11,TP53, and VHL, wherein an adapter sequence is ligated to the ends of theplurality of amplicons; and (c) detecting at least one mutation in atleast one of the plurality of amplicons using high throughput massiveparallel sequencing. In some embodiments, the dried biological fluidsample is dried plasma, dried serum, or dried whole blood. In someembodiments, the dried biological fluid sample comprises ananticoagulant (e.g., EDTA, heparin). In certain embodiments, the driedbiological fluid sample is obtained from a patient having, or issuspected of having a hereditary cancer. The hereditary cancer may bebreast cancer, ovarian cancer, colon cancer, or skin cancer.

Additionally or alternatively, in some embodiments, the dried biologicalfluid sample on the absorbent tip of the microsampling device iscollected from a patient via fingerstick. In certain embodiments, themicrosampling device is a MITRA® tip. Elution of the dried biologicalfluid sample may be performed by contacting the absorbent tip of themicrosampling device with a lysis buffer and Proteinase K. In certainembodiments, the lysis buffer comprises guanidine hydrochloride,Tris·Cl, EDTA, Tween 20, and Triton X-100. In a further embodiment, thelysis buffer comprises 800 mM guanidine hydrochloride; 30 mM Tris·Cl, pH8.0; 30 mM EDTA, pH 8.0; 5% Tween 20; and 0.5% Triton X-100. In otherembodiments, the lysis buffer comprises 2.5-10% sodium dodecyl sulphate.

Additionally or alternatively, in some embodiments, elution of the driedbiological fluid sample is performed by contacting the absorbent tip ofthe microsampling device with the lysis buffer for up to 15 minutes at90° C. Additionally or alternatively, in certain embodiments, elution ofthe dried biological fluid sample is performed by contacting theabsorbent tip of the microsampling device with Proteinase K for up to 1hour at 56° C. In other embodiments, elution of the dried biologicalfluid sample is performed by contacting the absorbent tip of themicrosampling device with Proteinase K for up to 16-18 hours at 56° C.In some embodiments, the sample volume of the microsampling device is nomore than 10-20 μL.

In some embodiments of the method, no more than 400 ng of genomic DNA iseluted from the absorbent tip of the microsampling device. In otherembodiments of the method, about 100 ng to about 400 ng of genomic DNAis eluted from the absorbent tip of the microsampling device.

In certain embodiments, the high throughput massive parallel sequencingis performed using pyrosequencing, reversible dye-terminator sequencing,SOLiD sequencing, Ion semiconductor sequencing, Helioscope singlemolecule sequencing, sequencing by synthesis, sequencing by ligation, orSMRT™ sequencing. In some embodiments of the method, the adaptersequence is a P5 adapter, P7 adapter, P1 adapter, A adapter, or IonXpress™ barcode adapter.

Additionally or alternatively, in some embodiments, the plurality ofamplicons further comprises a unique index sequence. In certainembodiments, the plurality of amplicons are enriched using a bait setcomprising nucleic acid sequences that are complementary to at least oneof the plurality of amplicons. In some embodiments, the nucleic acidsequences of the bait set are RNA baits, DNA baits, or a combinationthereof.

In another aspect, the present disclosure provides a method fordetecting at least one mutation in a plurality of hereditarycancer-related genes in a dried biological fluid sample comprisingisolating genomic DNA from a dried biological fluid sample eluted froman absorbent tip of a microsampling device with a lysis buffer andProteinase K, wherein the plurality of hereditary cancer-related genescomprises APC, ATM, BARD1, BMPR1A, BRCA1, BRCA2, BRIP1, CDH1, CDK4,CDKN2A (p14ARF and p16), CHEK2, EPCAM, MEN1, MLH1, MSH2, MSH6, MUTYH,NBN, NF1, PALB2, PMS2, POLD1, POLE, PTEN, RAD51C, RAD51D, RET, SDHB,SDHC, SDHD, SMAD4, STK11, TP53, and VHL. In certain embodiments, the atleast one mutation in the plurality of hereditary cancer-related genesis detected using high throughput massive parallel sequencing. In someembodiments, the lysis buffer comprises guanidine hydrochloride,Tris·Cl, EDTA, Tween 20, and Triton X-100.

In one aspect, the present disclosure provides a method for selecting apatient exhibiting cancer symptoms, or a patient at risk for hereditarycancer, for treatment with an anti-cancer therapeutic agent comprising(a) eluting a dried blood sample under conditions that result in therelease of genomic DNA from blood cells, wherein the dried blood sampleis collected from the patient with a volumetric absorptive microsamplingdevice; (b) isolating genomic DNA from the eluted dried blood sample;(c) generating a library comprising amplicons corresponding to each of aplurality of hereditary cancer-related genes comprising APC, ATM, BARD1,BMPR1A, BRCA1, BRCA2, BRIP1, CDH1, CDK4, CDKN2A (p14ARF and p16), CHEK2,EPCAM, MEN1, MLH1, MSH2, MSH6, MUTYH, NBN, NF1, PALB2, PMS2, POLD1,POLE, PTEN, RAD51C, RAD51D, RET, SDHB, SDHC, SDHD, SMAD4, STK11, TP53,and VHL, wherein an adapter sequence is ligated to the ends of theplurality of amplicons; (d) detecting at least one mutation in at leastone of the plurality of amplicons using high throughput massive parallelsequencing; and (e) selecting the patient for treatment with ananti-cancer therapeutic agent, if a mutation in at least one of theplurality of amplicons corresponding to one or more of APC, ATM, BARD1,BMPR1A, BRCA1, BRCA2, BRIP1, CDH1, CDK4, CDKN2A (p14ARF and p16), CHEK2,EPCAM MEN1, MLH1, MSH2, MSH6, MUTYH, NBN, NF1, PALB2, PMS2, POLD1, POLE,PTEN, RAD51C, RAD51D, RET, SDHB, SDHC, SDHD, SMAD4, STK11, TP53, and VHLis detected. In some embodiments, the volumetric absorptivemicrosampling device is a MITRA® tip. In certain embodiments, thepatient has, or is at risk for a hereditary cancer selected from thegroup consisting of breast cancer, ovarian cancer, skin cancer, or coloncancer.

In any of the above embodiments, the anti-cancer therapeutic agent isone or more agents selected from the group consisting ofcyclophosphamide, fluorouracil (or 5-fluorouracil or 5-FU),methotrexate, edatrexate (10-ethyl-10-deaza-aminopterin), thiotepa,carboplatin, cisplatin, taxanes, paclitaxel, protein-bound paclitaxel,docetaxel, vinorelbine, tamoxifen, raloxifene, toremifene, fulvestrant,gemcitabine, irinotecan, ixabepilone, temozolmide, topotecan,vincristine, vinblastine, eribulin, mutamycin, capecitabine,anastrozole, exemestane, letrozole, leuprolide, abarelix, buserlin,goserelin, megestrol acetate, risedronate, pamidronate, ibandronate,alendronate, denosumab, zoledronate, trastuzumab, tykerb,anthracyclines, bevacizumab, aldesleukin, cobimetinib, dabrafenib,dacarbazine, talimogene laherparepvec, imiquimod, recombinant InterferonAlfa-2b, ipilimumab, pembrolizumab, trametinib, nivolumab, peginterferonAlfa-2b, sonidegib, vismodegib, vemurafenib, cetuximab, irinotecanhydrochloride, leucovorin calcium, trifluridine and tipiracilhydrochloride, oxaliplatin, panitumumab, ramucirumab, regorafenib, andzivaflibercept.

Also disclosed herein are kits for detecting at least one mutation in aplurality of hereditary cancer-related genes in a dried biological fluidsample comprising a skin puncture tool, a volumetric absorptivemicrosampling device, a lysis buffer, and proteinase K, wherein theplurality of hereditary cancer-related genes comprises APC, ATM, BARD1,BMPR1A, BRCA1, BRCA2, BRIP1, CDH1, CDK4, CDKN2A (p14ARF and p16), CHEK2,EPCAM, MEN1, MLH1, MSH2, MSH6, MUTYH, NBN, NF1, PALB2, PMS2, POLD1,POLE, PTEN, RAD51C, RAD51D, RET, SDHB, SDHC, SDHD, SMAD4, STK11, TP53,and VHL. The lysis buffer may comprise guanidine hydrochloride, Tris·Cl,EDTA, Tween 20, and Triton X-100.

In some embodiments, the kits of the present technology further compriseone or more primer pairs that hybridize to one or more regions or exonsof one or more of the plurality of hereditary cancer-related genes.Additionally or alternatively, in some embodiments, the kits of thepresent technology further comprise one or more bait sequences thathybridize to one or more regions or exons of one or more of theplurality of hereditary cancer-related genes.

In any of the above embodiments of the kits of the present technology,the volumetric absorptive microsampling device is a MITRA® tip.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows DNA yield per MITRA® tip using the three differentextraction methods as described in Example 1.

FIG. 2(a) shows the minimum read coverage per target region obtainedfrom dried blood samples eluted from MITRA® tips on a 34-gene cancerpredisposition panel (MyVantage™ Hereditary Comprehensive Cancer Panel).FIG. 2(b) shows the minimum read coverage per target region with driedblood samples obtained using single or dual-MITRA® tip extraction on a34-gene cancer predisposition panel (MyVantage™ Hereditary ComprehensiveCancer Panel). “Ops” refers to routine operational samples.

FIG. 3 shows a comparison of DNA yields obtained from MITRA® tips whenusing different DNA extraction methods. ‘ON’ means the samples wereincubated with a particular lysis buffer overnight.

FIG. 4(a) shows the minimum read coverage per target region obtainedfrom dried blood samples eluted from a single MITRA® tip (DNA yieldsranging between 109-358 ng) on a 34-gene cancer predisposition panel(MyVantage™ Hereditary Comprehensive Cancer Panel). DNA extraction wasperformed using the QIAsymphony® platform. FIG. 4(b) shows the minimumread coverage per target region with dried blood samples obtained usingsingle or dual-MITRA ® tip extraction on a 34-gene cancer predispositionpanel (MyVantage™ Hereditary Comprehensive Cancer Panel). 4 samples weresubjected to dual-MITRA® tip extraction and resulted in DNA yieldsranging between 340-543 ng. 1 sample (AS) was subjected to single tipextraction and had a DNA yield of 214 ng.

FIG. 5 shows the effective amplification of 10 kb and 18 kb regionscorresponding to CHEK2 and PMS2 target regions respectively, vialong-range PCR when using dried blood samples eluted from MITRA® tips.

FIG. 6 shows the minimum read coverage of various CHEK2 and PMS2 exonsobtained from dried blood samples eluted from MITRA® tips.

FIG. 7 shows the minimum read coverage per target region using DNAobtained from (a) blood collected by MITRA® tips via fingerstick, (b)MITRA® tips wicked with EDTA whole blood, and (c) EDTA whole bloodobtained from conventional blood draws.

DETAILED DESCRIPTION

The present disclosure provides methods for determining whether apatient exhibiting cancer symptoms, or at risk for hereditary cancerssuch as breast cancer, ovarian cancer, colon cancer, or skin cancer,will benefit from treatment with one or more therapeutic agents. Thesemethods are based on detecting hereditary cancer-related mutations insmall-volume dried biological fluid samples that are collected using avolumetric absorptive microsampling device. Further, the methodsdisclosed herein retain their analytical sensitivity when used on driedbiological fluid samples containing anticoagulants, such as EDTA, whichare known PCR-inhibitors. See Huggett et al., BMC Res Notes. 1: 70(2008). Kits for use in practicing the methods are also provided.

Definitions

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the present technology belongs.

As used herein, the term “about” in reference to a number is generallytaken to include numbers that fall within a range of 1%-10% in eitherdirection (greater than or less than) of the number unless otherwisestated or otherwise evident from the context.

The term “adapter” refers to a short, chemically synthesized, nucleicacid sequence which can be used to ligate to the end of a nucleic acidsequence in order to facilitate attachment to another molecule. Theadapter can be single-stranded or double-stranded. An adapter canincorporate a short (typically less than 50 base pairs) sequence usefulfor PCR amplification or sequencing.

As used herein, the “administration” of a therapeutic agent or drug to asubject includes any route of introducing or delivering to a subject acompound to perform its intended function. Administration can be carriedout by any suitable route, including orally, intranasally, parenterally(intravenously, intramuscularly, intraperitoneally, or subcutaneously),or topically. Administration includes self-administration and theadministration by another.

As used herein, an “alteration” of a gene or gene product (e.g., amarker gene or gene product) refers to the presence of a mutation ormutations within the gene or gene product, e.g., a mutation, whichaffects the quantity or activity of the gene or gene product, ascompared to the normal or wild-type gene. The genetic alteration canresult in changes in the quantity, structure, and/or activity of thegene or gene product in a cancer tissue or cancer cell, as compared toits quantity, structure, and/or activity, in a normal or healthy tissueor cell (e.g., a control). For example, an alteration which isassociated with cancer can have an altered nucleotide sequence (e.g., amutation), amino acid sequence, chromosomal translocation,intra-chromosomal inversion, copy number, expression level, proteinlevel, protein activity, in a cancer tissue or cancer cell, as comparedto a normal, healthy tissue or cell. Exemplary mutations include, butare not limited to, point mutations (e.g., silent, missense, ornonsense), deletions, insertions, inversions, linking mutations,duplications, translocations, inter- and intra-chromosomalrearrangements. Mutations can be present in the coding or non-codingregion of the gene. In certain embodiments, the alterations areassociated with a phenotype, e.g., a cancerous phenotype (e.g., one ormore of cancer risk, cancer progression, cancer treatment or resistanceto cancer treatment).

As used herein, the terms “amplify” or “amplification” with respect tonucleic acid sequences, refer to methods that increase therepresentation of a population of nucleic acid sequences in a sample.Copies of a particular target nucleic acid sequence generated in vitroin an amplification reaction are called “amplicons” or “amplificationproducts”. Amplification may be exponential or linear. A target nucleicacid may be DNA (such as, for example, genomic DNA and cDNA) or RNA.While the exemplary methods described hereinafter relate toamplification using polymerase chain reaction (PCR), numerous othermethods such as isothermal methods, rolling circle methods, etc., arewell known to the skilled artisan. The skilled artisan will understandthat these other methods may be used either in place of, or togetherwith, PCR methods. See, e.g., Saiki, “Amplification of Genomic DNA” inPCR PROTOCOLS, Innis et al., Eds., Academic Press, San Diego, Calif.1990, pp 13-20; Wharam, et al., Nucleic Acids Res. 29(11):E54-E54(2001).

“Bait”, as used herein, is a type of hybrid capture reagent thatretrieves target nucleic acid sequences for sequencing. A bait can be anucleic acid molecule, e.g., a DNA or RNA molecule, which can hybridizeto (e.g., be complementary to), and thereby allow capture of a targetnucleic acid. In one embodiment, a bait is an RNA molecule (e.g., anaturally-occurring or modified RNA molecule); a DNA molecule (e.g., anaturally-occurring or modified DNA molecule), or a combination thereof.In other embodiments, a bait includes a binding entity, e.g., anaffinity tag, that allows capture and separation, e.g., by binding to abinding entity, of a hybrid formed by a bait and a nucleic acidhybridized to the bait. In one embodiment, a bait is suitable forsolution phase hybridization.

As used herein, “bait set” refers to one or a plurality of baitmolecules.

The terms “cancer” or “tumor” are used interchangeably and refer to thepresence of cells possessing characteristics typical of cancer-causingcells, such as uncontrolled proliferation, immortality, metastaticpotential, rapid growth and proliferation rate, and certaincharacteristic morphological features. Cancer cells are often in theform of a tumor, but such cells can exist alone within an animal, or canbe a non-tumorigenic cancer cell. As used herein, the term “cancer”includes premalignant, as well as malignant cancers.

The terms “complement”, “complementary” or “complementarity” as usedherein with reference to polynucleotides (i.e., a sequence ofnucleotides such as an oligonucleotide or a target nucleic acid) referto the Watson/Crick base-pairing rules. The complement of a nucleic acidsequence as used herein refers to an oligonucleotide which, when alignedwith the nucleic acid sequence such that the 5′ end of one sequence ispaired with the 3′ end of the other, is in “antiparallel association.”For example, the sequence “5′-A-G-T-3′” is complementary to the sequence“3′-T-C-A-5′.” Certain bases not commonly found in naturally-occurringnucleic acids may be included in the nucleic acids described herein.These include, for example, inosine, 7-deazaguanine, Locked NucleicAcids (LNA), and Peptide Nucleic Acids (PNA). Complementarity need notbe perfect; stable duplexes may contain mismatched base pairs,degenerative, or unmatched bases. Those skilled in the art of nucleicacid technology can determine duplex stability empirically considering anumber of variables including, for example, the length of theoligonucleotide, base composition and sequence of the oligonucleotide,ionic strength and incidence of mismatched base pairs. A complementsequence can also be an RNA sequence complementary to the DNA sequenceor its complement sequence, and can also be a cDNA.

The term “substantially complementary” as used herein means that twosequences hybridize under stringent hybridization conditions. Theskilled artisan will understand that substantially complementarysequences need not hybridize along their entire length. In particular,substantially complementary sequences may comprise a contiguous sequenceof bases that do not hybridize to a target sequence, positioned 3′ or 5′to a contiguous sequence of bases that hybridize under stringenthybridization conditions to a target sequence.

As used herein, a “control” is an alternative sample used in anexperiment for comparison purpose. A control can be “positive” or“negative.” A “control nucleic acid sample” or “reference nucleic acidsample” as used herein, refers to nucleic acid molecules from a controlor reference sample. In certain embodiments, the reference or controlnucleic acid sample is a wild type or a non-mutated DNA or RNA sequence.In certain embodiments, the reference nucleic acid sample is purified orisolated (e.g., it is removed from its natural state). In otherembodiments, the reference nucleic acid sample is from a non-tumorsample, e.g., a blood control, a normal adjacent tumor (NAT), or anyother non-cancerous sample from the same or a different subject.

As used herein, the term “detecting” refers to determining the presenceof a mutation or alteration in a nucleic acid of interest in a sample.Detection does not require the method to provide 100% sensitivity.

As used herein, the term “effective amount” refers to a quantitysufficient to achieve a desired therapeutic and/or prophylactic effect,e.g., an amount which results in the prevention of, or a decrease inhereditary cancer, or one or more symptoms associated with hereditarycancer. In the context of therapeutic or prophylactic applications, theamount of a therapeutic agent administered to the subject will depend onthe type and severity of the disease and on the characteristics of theindividual, such as general health, age, sex, body weight and toleranceto drugs. It will also depend on the degree, severity and type ofdisease. The skilled artisan will be able to determine appropriatedosages depending on these and other factors. As used herein, a“therapeutically effective amount” of a therapeutic drug or agent ismeant levels in which the physiological effects of a hereditary cancersuch as breast cancer, ovarian cancer, colon cancer, or skin cancer are,at a minimum, ameliorated. A therapeutically effective amount can begiven in one or more administrations.

As used herein, the terms “extraction” or “isolation” refer to anyaction taken to separate nucleic acids from other cellular materialpresent in the sample. The term extraction or isolation includesmechanical or chemical lysis, addition of detergent or protease, orprecipitation and removal of other cellular material.

“Gene” as used herein refers to a DNA sequence that comprises regulatoryand coding sequences necessary for the production of an RNA, which mayhave a non-coding function (e.g., a ribosomal or transfer RNA) or whichmay include a polypeptide or a polypeptide precursor. The RNA orpolypeptide may be encoded by a full length coding sequence or by anyportion of the coding sequence so long as the desired activity orfunction is retained. Although a sequence of the nucleic acids may beshown in the form of DNA, a person of ordinary skill in the artrecognizes that the corresponding RNA sequence will have a similarsequence with the thymine being replaced by uracil, i.e., “T” isreplaced with “U.”

The term “hybridize” as used herein refers to a process where twosubstantially complementary nucleic acid strands (at least about 65%complementary over a stretch of at least 14 to 25 nucleotides, at leastabout 75%, or at least about 90% complementary) anneal to each otherunder appropriately stringent conditions to form a duplex orheteroduplex through formation of hydrogen bonds between complementarybase pairs. Hybridizations are typically and preferably conducted withprobe-length nucleic acid molecules, preferably 15-100 nucleotides inlength, more preferably 18-50 nucleotides in length. Nucleic acidhybridization techniques are well known in the art. See, e.g., Sambrook,et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition,Cold Spring Harbor Press, Plainview, N.Y. Hybridization and the strengthof hybridization (i.e., the strength of the association between thenucleic acids) is influenced by such factors as the degree ofcomplementarity between the nucleic acids, stringency of the conditionsinvolved, and the thermal melting point (T_(m)) of the formed hybrid.Those skilled in the art understand how to estimate and adjust thestringency of hybridization conditions such that sequences having atleast a desired level of complementarity will stably hybridize, whilethose having lower complementarity will not. For examples ofhybridization conditions and parameters, see, e.g., Sambrook, et al.,1989, Molecular Cloning: A Laboratory Manual, Second Edition, ColdSpring Harbor Press, Plainview, N.Y.; Ausubel, F. M. et al. 1994,Current Protocols in Molecular Biology, John Wiley & Sons, Secaucus,N.J. In some embodiments, specific hybridization occurs under stringenthybridization conditions. An oligonucleotide or polynucleotide (e.g., aprobe or a primer) that is specific for a target nucleic acid will“hybridize” to the target nucleic acid under suitable conditions.

As used herein, the terms “individual”, “patient”, or “subject” can bean individual organism, a vertebrate, a mammal, or a human. In apreferred embodiment, the individual, patient or subject is a human.

As used herein, the term “library” refers to a collection of nucleicacid sequences, e.g., a collection of nucleic acids derived from wholegenomic, subgenomic fragments, cDNA, cDNA fragments, RNA, RNA fragments,or a combination thereof. In one embodiment, a portion or all of thelibrary nucleic acid sequences comprises an adapter sequence. Theadapter sequence can be located at one or both ends. The adaptersequence can be useful, e.g., for a sequencing method (e.g., an NGSmethod), for amplification, for reverse transcription, or for cloninginto a vector.

The library can comprise a collection of nucleic acid sequences, e.g., atarget nucleic acid sequence (e.g., a tumor nucleic acid sequence), areference nucleic acid sequence, or a combination thereof). In someembodiments, the nucleic acid sequences of the library can be derivedfrom a single subject. In other embodiments, a library can comprisenucleic acid sequences from more than one subject (e.g., 2, 3, 4, 5, 6,7, 8, 9, 10, 20, 30 or more subjects). In some embodiments, two or morelibraries from different subjects can be combined to form a libraryhaving nucleic acid sequences from more than one subject. In oneembodiment, the subject is human having, or at risk of having, ahereditary cancer.

A “library nucleic acid sequence” refers to a nucleic acid molecule,e.g., a DNA, RNA, or a combination thereof, that is a member of alibrary. Typically, a library nucleic acid sequence is a DNA molecule,e.g., genomic DNA or cDNA. In some embodiments, a library nucleic acidsequence is fragmented, e.g., sheared or enzymatically prepared, genomicDNA. In certain embodiments, the library nucleic acid sequences comprisesequence from a subject and sequence not derived from the subject, e.g.,adapter sequence, a primer sequence, or other sequences that allow foridentification, e.g., “barcode” sequences.

“Next generation sequencing or NGS” as used herein, refers to anysequencing method that determines the nucleotide sequence of eitherindividual nucleic acid molecules (e.g., in single molecule sequencing)or clonally expanded proxies for individual nucleic acid molecules in ahigh throughput parallel fashion (e.g., greater than 10³, 10⁴, 10⁵ ormore molecules are sequenced simultaneously). In one embodiment, therelative abundance of the nucleic acid species in the library can beestimated by counting the relative number of occurrences of theircognate sequences in the data generated by the sequencing experiment.Next generation sequencing methods are known in the art, and aredescribed, e.g., in Metzker, M. Nature Biotechnology Reviews 11:31-46(2010).

As used herein, “oligonucleotide” refers to a molecule that has asequence of nucleic acid bases on a backbone comprised mainly ofidentical monomer units at defined intervals. The bases are arranged onthe backbone in such a way that they can bind with a nucleic acid havinga sequence of bases that are complementary to the bases of theoligonucleotide. The most common oligonucleotides have a backbone ofsugar phosphate units. A distinction may be made betweenoligodeoxyribonucleotides that do not have a hydroxyl group at the 2′position and oligoribonucleotides that have a hydroxyl group at the 2′position. Oligonucleotides may also include derivatives, in which thehydrogen of the hydroxyl group is replaced with organic groups, e.g., anallyl group. Oligonucleotides that function as primers or probes aregenerally at least about 10-15 nucleotides in length or up to about 70,100, 110, 150 or 200 nucleotides in length, and more preferably at leastabout 15 to 25 nucleotides in length. Oligonucleotides used as primersor probes for specifically amplifying or specifically detecting aparticular target nucleic acid generally are capable of specificallyhybridizing to the target nucleic acid.

As used herein, the term “primer” refers to an oligonucleotide, which iscapable of acting as a point of initiation of nucleic acid sequencesynthesis when placed under conditions in which synthesis of a primerextension product which is complementary to a target nucleic acid strandis induced, i.e., in the presence of different nucleotide triphosphatesand a polymerase in an appropriate buffer (“buffer” includes pH, ionicstrength, cofactors etc.) and at a suitable temperature. One or more ofthe nucleotides of the primer can be modified for instance by additionof a methyl group, a biotin or digoxigenin moiety, a fluorescent tag orby using radioactive nucleotides. A primer sequence need not reflect theexact sequence of the template. For example, a non-complementarynucleotide fragment may be attached to the 5′ end of the primer, withthe remainder of the primer sequence being substantially complementaryto the strand. The term primer as used herein includes all forms ofprimers that may be synthesized including peptide nucleic acid primers,locked nucleic acid primers, phosphorothioate modified primers, labeledprimers, and the like. The term “forward primer” as used herein means aprimer that anneals to the anti-sense strand of double-stranded DNA(dsDNA). A “reverse primer” anneals to the sense-strand of dsDNA.

Primers are typically at least 10, 15, 18, or 30 nucleotides in lengthor up to about 100, 110, 125, or 200 nucleotides in length. In someembodiments, primers are preferably between about 15 to about 60nucleotides in length, and most preferably between about 25 to about 40nucleotides in length. In some embodiments, primers are 15 to 35nucleotides in length. There is no standard length for optimalhybridization or polymerase chain reaction amplification. An optimallength for a particular primer application may be readily determined inthe manner described in H. Erlich, PCR Technology, PRINCIPLES ANDAPPLICATION FOR DNA AMPLIFICATION, (1989).

As used herein, the term “primer pair” refers to a forward and reverseprimer pair (i.e., a left and right primer pair) that can be usedtogether to amplify a given region of a nucleic acid of interest.

“Probe” as used herein refers to nucleic acid that interacts with atarget nucleic acid via hybridization. A probe may be fullycomplementary to a target nucleic acid sequence or partiallycomplementary. The level of complementarity will depend on many factorsbased, in general, on the function of the probe. Probes can be labeledor unlabeled, or modified in any of a number of ways well known in theart. A probe may specifically hybridize to a target nucleic acid. Probesmay be DNA, RNA or a RNA/DNA hybrid. Probes may be oligonucleotides,artificial chromosomes, fragmented artificial chromosome, genomicnucleic acid, fragmented genomic nucleic acid, RNA, recombinant nucleicacid, fragmented recombinant nucleic acid, peptide nucleic acid (PNA),locked nucleic acid, oligomer of cyclic heterocycles, or conjugates ofnucleic acid. Probes may comprise modified nucleobases, modified sugarmoieties, and modified internucleotide linkages. Probes are typically atleast about 10, 15, 20, 25, 30, 35, 40, 50, 60, 75, 100 nucleotides ormore in length.

As used herein, the term “sample” refers to clinical samples obtainedfrom a patient. In preferred embodiments, a sample is obtained from abiological source (i.e., a “biological sample”), such as tissue orbodily fluid collected from a subject. Sample sources include, but arenot limited to, mucus, sputum (processed or unprocessed), bronchialalveolar lavage (BAL), bronchial wash (BW), blood, bodily fluids,cerebrospinal fluid (CSF), urine, plasma, serum, or tissue (e.g., biopsymaterial). Preferred sample sources include plasma, serum, or wholeblood.

The term “sensitivity,” as used herein in reference to the methods ofthe present technology, is a measure of the ability of a method todetect a preselected sequence variant in a heterogeneous population ofsequences. A method has a sensitivity of S % for variants of F if, givena sample in which the preselected sequence variant is present as atleast F % of the sequences in the sample, the method can detect thepreselected sequence at a preselected confidence of C %, S % of thetime. By way of example, a method has a sensitivity of 90% for variantsof 5% if, given a sample in which the preselected variant sequence ispresent as at least 5% of the sequences in the sample, the method candetect the preselected sequence at a preselected confidence of 99%, 9out of 10 times (F=5%; C=99%; S=90%). Exemplary sensitivities include atleast 50, 60, 70, 80, 90, 95, 98, and 99%.

The term “specific” as used herein in reference to an oligonucleotideprimer means that the nucleotide sequence of the primer has at least 12bases of sequence identity with a portion of the nucleic acid to beamplified when the oligonucleotide and the nucleic acid are aligned. Anoligonucleotide primer that is specific for a nucleic acid is one that,under the stringent hybridization or washing conditions, is capable ofhybridizing to the target of interest and not substantially hybridizingto nucleic acids which are not of interest. Higher levels of sequenceidentity are preferred and include at least 75%, at least 80%, at least85%, at least 90%, at least 85-95% and more preferably at least 98%sequence identity. Sequence identity can be determined using acommercially available computer program with a default setting thatemploys algorithms well known in the art. As used herein, sequences thathave “high sequence identity” have identical nucleotides at least atabout 50% of aligned nucleotide positions, preferably at least at about60% of aligned nucleotide positions, and more preferably at least atabout 75% of aligned nucleotide positions.

“Specificity,” as used herein, is a measure of the ability of a methodto distinguish a truly occurring preselected sequence variant fromsequencing artifacts or other closely related sequences. It is theability to avoid false positive detections. False positive detectionscan arise from errors introduced into the sequence of interest duringsample preparation, sequencing error, or inadvertent sequencing ofclosely related sequences like pseudo-genes or members of a gene family.A method has a specificity of X % if, when applied to a sample set ofN_(Total) sequences, in which X_(True) sequences are truly variant andX_(Not true) are not truly variant, the method selects at least X % ofthe not truly variant as not variant. E.g., a method has a specificityof 90% if, when applied to a sample set of 1,000 sequences, in which 500sequences are truly variant and 500 are not truly variant, the methodselects 90% of the 500 not truly variant sequences as not variant.Exemplary specificities include at least 50, 60, 70, 80, 90, 95, 98, and99%.

The term “stringent hybridization conditions” as used herein refers tohybridization conditions at least as stringent as the following:hybridization in 50% formamide, 5× SSC, 50 mM NaH₂PO4, pH 6.8, 0.5% SDS,0.1 mg/mL sonicated salmon sperm DNA, and 5× Denhart's solution at 42°C. overnight; washing with 2× SSC, 0.1% SDS at 45° C.; and washing with0.2× SSC, 0.1% SDS at 45° C. In another example, stringent hybridizationconditions should not allow for hybridization of two nucleic acids whichdiffer over a stretch of 20 contiguous nucleotides by more than twobases.

The terms “target nucleic acid” or “target sequence” as used hereinrefer to a nucleic acid sequence of interest to be detected and/orquantified in the sample to be analyzed. Target nucleic acid may becomposed of segments of a chromosome, a complete gene with or withoutintergenic sequence, segments or portions of a gene with or withoutintergenic sequence, or sequence of nucleic acids which probes orprimers are designed. Target nucleic acids may include a wild-typesequence(s), a mutation, deletion, insertion or duplication, tandemrepeat elements, a gene of interest, a region of a gene of interest orany upstream or downstream region thereof. Target nucleic acids mayrepresent alternative sequences or alleles of a particular gene. Targetnucleic acids may be derived from genomic DNA, cDNA, or RNA.

As used herein, the terms “treat,” “treating” or “treatment” refer, toan action to obtain a beneficial or desired clinical result including,but not limited to, alleviation or amelioration of one or more signs orsymptoms of a disease or condition (e.g., regression, partial orcomplete), diminishing the extent of disease, stability (i.e., notworsening, achieving stable disease) state of disease, amelioration orpalliation of the disease state, diminishing rate of or time toprogression, and remission (whether partial or total).

Microsampling Devices Employed in the Methods of the Present Technology

Conventional dried blood spotting techniques are accompanied by a numberof drawbacks, including imprecise sample volume and reliance on aconstant sample viscosity (i.e., the expectation that the sample willspread uniformly on the sample card). A constant viscosity results inblood spot diameters remaining constant when equal volume samples areadministered to the cards. However, viscosity varies significantlybetween blood samples because of differing hematocrit (HCT) or packedcell volume (PCV) levels in the blood. Samples with high hematocritlevels form smaller diameter spots on the bloodspot papers, leading todifferent concentrations of blood within the fixed diameter of the spotssampled. PCV levels are believed to show a variance of about 45% in spotdiameters. As internal standards are sprayed onto the spotted blood,this can result in a 45% error in quantitation. The microsamplingdevices employed in the methods disclosed herein confer severaladvantages, including the collection of more precise blood volumes, lackof hematocrit bias, and the ability to be easily automated with standardliquid handlers for lab processing.

Additionally, conventional blood spot techniques require a comparativelylarge volume of blood relative to the disclosed microsampling devices. Adried blood spot would generally require 50-75 μl per spot, while amicrosampling device can yield results from approximately 20 μl. It hasbeen recognized in the art that dried blood spots often have performancevariability issues for detecting viral load compared to other samplestypes, such as plasma (Pannus et al., Medicine, 95:48(e5475) (2016)),and the volume of a dried blood spot may need to be significantly higherfor certain types of assessment (e.g., optical density) compared toother sample types, such as serum (Brandao et al., J. Clin. Virol.,57:98-102 (2013)). Indeed, found that using both dried blood spot andplasma spot screening for detecting viral load and treatment failure inHIV patients receiving antiretroviral therapy found that both yielded ahigh rate of false positives (Sawadogo et al., J. Clin. Microbiol.,52(11):3878-83 (2014)).

The microsampling device useful in the methods of the present technologycomprises an absorbent tip having a distal end and a proximal end. Thewidth of the distal end of the absorbent tip is narrow compared to thewidth of the proximal end. The proximal end is attached to a holder,whereas the distal end is configured to contact a fluid to be absorbed,such as blood. The microsampling device permits biological fluidsamples, such as blood, to be easily dried, shipped, and then lateranalyzed. In certain embodiments, the biological fluid is blood from afingerstick.

Wicking action draws the blood into the absorbent tip. An optionalbarrier between the absorbent tip and the holder prevents blood frompassing or wicking to the holder. The absorbent tip is composed of amaterial that wicks up substantially the same volume of fluid even whenexcess fluid is available (volumetric absorptive microsampling orVAMS™). The volume of the absorbent tip affects the volume of fluidabsorbed. The size and shape of the absorbent tip may be varied toadjust the volume of absorbed blood and the rate of absorption. Bloodvolumes of about 7-15 μL, about 20 μL and even up to about 30 L may beacceptable. The sampling time may be about 2 seconds, about 3 seconds,about 5 seconds, or up to about 10 seconds.

In some embodiments, the material used for the absorbent tip ishydrophilic (e.g., polyester). Alternatively, the material may initiallybe hydrophobic and is subsequently treated to make it hydrophilic.Hydrophobic matrices may be rendered hydrophilic by a variety of knownmethods, such as plasma treatment or surfactant treatment (e.g.,Tween-40 or Tween-80) of the matrix. In some embodiments, plasmatreatment is used to render a hydrophobic material such as polyolefin,e.g., polyethylene, hydrophilic. Alternatively, the grafting ofhydrophilic polymers to the surface and the chemical functionalizationof active groups on the surface with polar or hydrophilic molecules suchas sugars can be used to achieve a hydrophilic surface for the absorbenttip. Covalent modification could also be used to add polar orhydrophilic functional groups to the surface of absorbent tip. Othersuitable materials for the absorbent tip include sintered glass,sintered steel, sintered ceramics, and sintered polymers of plastic, andsintered polyethylene.

In some embodiments, the microsampling device comprises an absorbent tipmade of a hydrophilic polymeric material of sufficient size to absorb amaximum of about 20 μL of blood in about 2-5 seconds, and having alength of less than about 5 mm (0.2 inches) and a cross-sectional areaof less than about 20 mm² and a density of less than about 4 g/cc. Insome embodiments, the absorbent tips are composed of polyethylene andconfigured to absorb about 1-20 microliters of blood, preferably within1-7 seconds, and more preferably within about 1-5 seconds. The absorbenttip may contain one or more of dried blood, dried anticoagulant or aninternal standard.

In certain embodiments, the absorbent tips have a volume of about 35mm³, absorb about 13-14 microliters of blood in about 3 seconds, absorb9-10 microliters of blood in about 2.5 seconds, and have a pore volumeof about 38%. In other embodiments, the absorbent tips have a volume ofabout 24 microliters, a density of about 0.6 g/cc, absorb about 10microliters of blood in about 2.5 seconds, and have a pore volume ofabout 40%. In some embodiments, the volumetric absorptive microsamplingdevice is a MITRA® tip, as described in US 2013/0116597, which is hereinincorporated by reference in its entirety.

The absorbent tip may be shaped with an exterior resembling a truncatedcone with a narrow and rounded distal end. In some embodiments, theholder has a cylindrical post that fits into a recess inside the centerof the absorbent tip and extending along the longitudinal axis of theabsorbent tip and holder. The conical shape of the absorbent tip helpswick the sample quickly and uniformly.

The holder may be adapted for use with a pipette. In some embodiments, atubular, conical shaped holder is preferred, with the absorbent tip onthe narrow end of the holder. The wider opposite end of the holder maybe closed, or open and hollow, and may optionally be configured toattach to a pipette tip. The holder may have outwardly extending flangesthat are arranged to abut mating structures in holders, drying racks ortest equipment to help position the absorbent tip at desired locationsin such holders, drying racks and test equipment.

In certain embodiments, the holder may include a pipette tip or atapering, tubular structure configured to nest with a pipette tip. Theabsorbent tip may be composed of polyethylene, and both the absorbenttip and holder are made under aseptic conditions, or are terminallysterilized. The absorbent tip may contain dried anti-coagulant. In someembodiments, the holder has a plurality of ribs extending along a lengthof the holder. The ribs may have a height and length selected to keepthe absorbent tip from contacting walls of a recess into which theholder and absorbent tip are placed for shipment, or for extraction ofthe dried blood in the absorbent tip.

After absorbing a small-volume sample, the absorbent tip is then dried.In some embodiments, the small-volume blood sample is dried for at least10 minutes, at least 20 minutes, at least 30 minutes, at least 40minutes, at least 50 minutes, at least 1 hour, at least 2 hours, atleast 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, atleast 8 hours, at least 12 hours, at least 16 hours, at least 20 hours,at least 24 hours, at least 48 hours, at least 72 hours, or at least 96hours at ambient or room temperature. In certain embodiments, thesmall-volume blood sample is dried for about 2-3 hours.

Drying can be done on a suitable rack or holder, or preferably theabsorbent tip and holder can be transferred to a special dryingcontainer configured to facilitate drying while minimizing contactbetween the absorbent tip and the walls of the drying container or otherpotential contaminant surfaces. The drying container may have adesiccant to facilitate drying. The drying container may also provide aprotective cover which may be sealed for transport to preventcontamination. In some embodiments, the cover has a surface onto whichprinted indicia may be written to identify the source of the dried bloodsample and provide other relevant information. In some embodiments, thedimensions of the container, and the relative positions of the holderswithin the container, will conform to SBS Microwell platespecifications. The microsampling device and the drying container may beplaced in a plastic bag along with a desiccant to assist with drying andcan either be shipped in this fashion, or shipped after the desiccant isremoved.

In some embodiments, the wider opposite end of the holder is hollow andthe container has a first portion with a mounting projection portionsized to fit into and releasably engage the hollow end of the holder.Additionally or alternatively, the container has a second portionreleasably fastened to the first portion and has a recess configured toenclose a portion of the holder for transportation of the holder. Thecontainer may comprise a plurality of openings allowing air to accessthe absorbent tip of the microsampling device. Moreover, the firstportion may have a side with an access port therein of sufficient sizeand located so that indicia may be applied through the port and onto theholder when the holder is on the mounting projection.

Upon receipt at the testing location, the absorbent tip may be eluted ina predetermined volume of a suitable buffer (as described herein) eithermanually or via automated means to extract the nucleic acids or proteinsof interest from dried blood. Physical agitation techniques such assonication or vortexing of the fluid and/or the absorbent tip mayaccelerate the extraction process from the dried blood into a liquidsample matrix. Physical separation techniques such as centrifugation,evaporation/reconstitution, concentration, precipitation, liquid/liquidextraction, and solid phase extraction can be used to further simplifythe sample matrix for further analysis.

Each container may enclose a plurality of holders, wherein each holdercomprises an absorbent tip at its distal end and has a hollow proximalend. The container likewise has a plurality of elongated mountingprojections each sized to fit into and releasably engage the hollow endsof the plurality of holders. The second portion of the container hasrecesses configured to separately enclose each of the plurality ofholders in a separate enclosure within the container. In certainembodiments, each of the plurality of holders has a plurality of ribsextending along a length of the holder with the ribs configured to keepthe absorbent tip from contacting walls of the container. As desired, adesiccant may be placed inside the container to help dry the blood inthe absorbent tip or maintain dryness. Each holder may have visibleindicia associating the holder with the container and with at least oneother holder, such as serial numbers with various portions of the numberindicating related holders/absorbent tips and the container in which theholders are shipped.

Nucleic Acid Extraction

In one aspect, the present disclosure provides a method for extractinggenomic DNA from a dried biological fluid sample collected with avolumetric absorptive microsampling device (e.g., MITRA® Tip). In someembodiments, the dried biological fluid sample is eluted by contactingthe absorbent tip of a volumetric absorptive microsampling device with alysis buffer and proteinase K. The lysis buffer may comprise guanidinehydrochloride, Tris·Cl, EDTA, Tween 20, and Triton X-100. Proteinase Kis a broad spectrum serine protease that is stable over a wide pH range(4-12), with a pH optimum of pH 8.0. The predominant site of ProteinaseK cleavage is the peptide bond adjacent to the carboxyl group ofaliphatic and aromatic amino acids with blocked alpha amino groups.Elevating the reaction temperature from 37° C. to 50-60° C. may increasethe Proteinase K activity by several fold. Proteinase K activity can beenhanced by the addition of 0.5-1% sodium dodecyl sulfate (SDS), 3 MGuanidinium chloride, 1 M Guanidinium thiocyanate, or 4 M urea.

Alternatively, other protocols for nucleic acid extraction may be usedin the methods of the present technology. Examples of other commerciallyavailable nucleic acid purification kits include Molzym GmbH & Co KG(Bremen, DE), Qiagen (Hilden, DE), Macherey-Nagel (Düren, DE), Roche(Basel, CH) or Sigma (Deisenhofen, DE). Other systems for nucleic acidpurification, which are based on the use of polystyrene beads etc., assupport material may also be used.

In some embodiments, extraction of genomic DNA from a dried biologicalfluid sample collected with a volumetric absorptive microsampling devicecomprises denaturing nucleoprotein complexes in cells present in thedried biological fluid sample. In certain embodiments, extraction ofgenomic DNA from a dried biological fluid sample collected with avolumetric absorptive microsampling device comprises removing proteincontaminants, inactivating nuclease activity, and/or removing biologicaland/or chemical contaminants present in the dried biological fluidsample.

In some embodiments, extraction of genomic DNA from a dried biologicalfluid sample collected with a volumetric absorptive microsampling devicemay be performed using automated DNA extraction platforms. In someembodiments, the automated DNA extraction platform has high-throughputcapacity, such as up to 100 extractions per cycle. In certainembodiments, extraction of genomic DNA from a dried biological fluidsample collected with a volumetric absorptive microsampling device maybe performed using commercially available automated workstations, suchas the QIAsymphony® or Hamilton® automation. In some embodiments,extraction of genomic DNA from a dried biological fluid sample collectedwith a volumetric absorptive microsampling device is performed on aBiorobot° EZ1™ automated system. In some embodiments, extraction ofgenomic DNA from a dried biological fluid sample collected with avolumetric absorptive microsampling device is performed usingcommercially available reagent kits.

Multiplex Ligation-dependent Probe Amplification (MLPA)

Multiplex ligation-dependent probe amplification (MLPA) is a variationof the multiplex polymerase chain reaction that permits amplification ofmultiple targets with only a single primer pair. The MLPA reaction canbe divided in four major steps: 1) DNA denaturation and hybridization ofMLPA hemi-probes; 2) ligation reaction; 3) PCR amplification; 4)separation of amplification products by electrophoresis. During thefirst step, the DNA is denatured and incubated overnight with a mixtureof MLPA probes. Each MLPA probe consists of two oligonucleotides (orhemi-probes) which recognize adjacent target sites on the DNA. Onehemi-probe oligonucleotide comprises a sequence recognized by theforward primer, while the other hemi-probe comprises a sequencerecognized by the reverse primer. The hemi-probes are ligated into acomplete probe only when both hemi-probe oligonucleotides are hybridizedto their respective targets. The advantage of using hemi-probes is thatonly the ligated oligonucleotides, but not the unbound hemi-probeoligonucleotides, are amplified. Because only ligated probes will beexponentially amplified during the subsequent PCR reaction, the numberof probe ligation products is a measure for the number of targetsequences in the sample.

Each complete probe has a unique length, so that its resulting ampliconscan be separated and identified by (capillary) electrophoresis. Thisfeature avoids the resolution limitations of multiplex PCR. Because theforward primer used for probe amplification is detectably labeled, eachamplicon generates a fluorescent peak which can be detected by acapillary sequencer. Comparing the peak pattern obtained on a givensample with that obtained on various reference samples, the relativequantity of each amplicon can be determined. This ratio is a measure forthe ratio in which the target sequence is present in the sample DNA.

NGS Platforms

Following the production of an adapter tagged amplicon library, theamplicons are sequenced using high throughput, massively parallelsequencing (i.e., next generation sequencing). In some embodiments, highthroughput, massively parallel sequencing employssequencing-by-synthesis with reversible dye terminators. In otherembodiments, sequencing is performed via sequencing-by-ligation. Inother embodiments, sequencing is single molecule sequencing. Examples ofNext Generation Sequencing techniques include, but are not limited topyrosequencing, Reversible dye-terminator sequencing, SOLiD sequencing,Ion semiconductor sequencing, Helioscope single molecule sequencing etc.

The Ion Torrent™ (Life Technologies, Carlsbad, Calif.) ampliconsequencing system employs a flow-based approach that detects pH changescaused by the release of hydrogen ions during incorporation ofunmodified nucleotides in DNA replication. For use with this system, asequencing library is initially produced by generating DNA fragmentsflanked by sequencing adapters. In some embodiments, these fragments canbe clonally amplified on particles by emulsion PCR. The particles withthe amplified template are then placed in a silicon semiconductorsequencing chip. During replication, the chip is flooded with onenucleotide after another, and if a nucleotide complements the DNAmolecule in a particular microwell of the chip, then it will beincorporated. A proton is naturally released when a nucleotide isincorporated by the polymerase in the DNA molecule, resulting in adetectable local change of pH. The pH of the solution then changes inthat well and is detected by the ion sensor. If homopolymer repeats arepresent in the template sequence, multiple nucleotides will beincorporated in a single cycle. This leads to a corresponding number ofreleased hydrogens and a proportionally higher electronic signal.

The 454™ GS FLX™ sequencing system (Roche, Germany), employs alight-based detection methodology in a large-scale parallelpyrosequencing system. Pyrosequencing uses DNA polymerization, addingone nucleotide species at a time and detecting and quantifying thenumber of nucleotides added to a given location through the lightemitted by the release of attached pyrophosphates. For use with the454198 system, adapter-ligated DNA fragments are fixed to smallDNA-capture beads in a water-in-oil emulsion and amplified by PCR(emulsion PCR). Each DNA-bound bead is placed into a well on a picotiterplate and sequencing reagents are delivered across the wells of theplate. The four DNA nucleotides are added sequentially in a fixed orderacross the picotiter plate device during a sequencing run. During thenucleotide flow, millions of copies of DNA bound to each of the beadsare sequenced in parallel. When a nucleotide complementary to thetemplate strand is added to a well, the nucleotide is incorporated ontothe existing DNA strand, generating a light signal that is recorded by aCCD camera in the instrument.

Sequencing technology based on reversible dye-terminators: DNA moleculesare first attached to primers on a slide and amplified so that localclonal colonies are formed. Four types of reversible terminator bases(RT-bases) are added, and non-incorporated nucleotides are washed away.Unlike pyrosequencing, the DNA can only be extended one nucleotide at atime. A camera takes images of the fluorescently labeled nucleotides,then the dye along with the terminal 3′ blocker is chemically removedfrom the DNA, allowing the next cycle.

Helicos Biosciences Corp's (Cambridge, Mass.) single-molecule sequencinguses DNA fragments with added polyA tail adapters, which are attached tothe flow cell surface. At each cycle, DNA polymerase and a singlespecies of fluorescently labeled nucleotide are added, resulting intemplate-dependent extension of the surface-immobilized primer-templateduplexes. The reads are performed by the Helioscope sequencer. Afteracquisition of images tiling the full array, chemical cleavage andrelease of the fluorescent label permits the subsequent cycle ofextension and imaging.

Sequencing by synthesis (SBS), like the “old style” dye-terminationelectrophoretic sequencing, relies on incorporation of nucleotides by aDNA polymerase to determine the base sequence. A DNA library withaffixed adapters is denatured into single strands and grafted to a flowcell, followed by bridge amplification to form a high-density array ofspots onto a glass chip. Reversible terminator methods use reversibleversions of dye-terminators, adding one nucleotide at a time, detectingfluorescence at each position by repeated removal of the blocking groupto allow polymerization of another nucleotide. The signal of nucleotideincorporation can vary with fluorescently labeled nucleotides,phosphate-driven light reactions and hydrogen ion sensing having allbeen used. Examples of SBS platforms include Illumina Ga., HiSeq 2500,HiSeq 1500, HiSeq 2000, or HiSeq 1000. The MiSeq® personal sequencingsystem (Illumina, Inc.) also employs sequencing by synthesis withreversible terminator chemistry.

In contrast to the sequencing by synthesis method, the sequencing byligation method uses a DNA ligase to determine the target sequence. Thissequencing method relies on enzymatic ligation of oligonucleotides thatare adjacent through local complementarity on a template DNA strand.This technology employs a partition of all possible oligonucleotides ofa fixed length, labeled according to the sequenced position.Oligonucleotides are annealed and ligated and the preferential ligationby DNA ligase for matching sequences results in a dinucleotide encodedcolor space signal at that position (through the release of afluorescently labeled probe that corresponds to a known nucleotide at aknown position along the oligo). This method is primarily used by LifeTechnologies' SOLiD™ sequencers. Before sequencing, the DNA is amplifiedby emulsion PCR. The resulting beads, each containing only copies of thesame DNA molecule, are deposited on a solid planar substrate.

SMRT™ sequencing is based on the sequencing by synthesis approach. TheDNA is synthesized in zero-mode wave-guides (ZMWs)-small well-likecontainers with the capturing tools located at the bottom of the well.The sequencing is performed with use of unmodified polymerase (attachedto the ZMW bottom) and fluorescently labeled nucleotides flowing freelyin the solution. The wells are constructed in a way that only thefluorescence occurring at the bottom of the well is detected. Thefluorescent label is detached from the nucleotide at its incorporationinto the DNA strand, leaving an unmodified DNA strand.

High-throughput sequencing of DNA can also take place using AnyDot-chips(Genovoxx, Germany), which allows monitoring of biological processes(e.g., miRNA expression or allele variability (SNP detection)). Forexample, the AnyDot-chips allow for 10×-50× enhancement of nucleotidefluorescence signal detection. Other high-throughput sequencing systemsinclude those disclosed in Venter, J., et al., Science 16 February 2001;Adams, M. et al., Science 24 March 2000; and M. J, Levene, et al.,Science 299:682-686, January 2003; as well as U.S. Application Pub. No.2003/0044781 and 2006/0078937.

Hereditary Cancer Detection Assays of the Present Technology

Provided herein are methods for detecting at least one mutation in aplurality of hereditary cancer-related genes in a dried biological fluidsample comprising isolating genomic DNA from a dried biological fluidsample eluted from an absorbent tip of a microsampling device (e.g.,MITRA® Tip) with a lysis buffer and Proteinase K, wherein the pluralityof hereditary cancer-related genes comprises APC, ATM, BARD1, BMPR1A,BRCA1, BRCA2, BRIP1, CDH1, CDK4, CDKN2A (p14ARF and p16), CHEK2, EPCAM,MEN1, MLH1, MSH2, MSH6, MUTYH, NBN, NF1, PALB2, PMS2, POLD1, POLE, PTEN,RAD51C, RAD51D, RET, SDHB, SDHC, SDHD, SMAD4, STK11, TP53, and VHL. Incertain embodiments, the at least one mutation in the plurality ofhereditary cancer-related genes is detected using high throughputmassive parallel sequencing. In some embodiments, the lysis buffercomprises guanidine hydrochloride, Tris·Cl, EDTA, Tween 20, and TritonX-100.

In one aspect, the present technology provides a method for detecting atleast one mutation in a plurality of hereditary cancer-related genes ina dried biological fluid sample comprising (a) extracting genomic DNAfrom a dried biological fluid sample eluted from an absorbent tip of amicrosampling device; (b) generating a library comprising ampliconscorresponding to each of the plurality of hereditary cancer-relatedgenes, said plurality of hereditary cancer-related genes comprising APC,ATM, BARD1, BMPR1A, BRCA1, BRCA2, BRIP1, CDH1, CDK4, CDKN2A (p14ARF andp16), CHEK2, EPCAM MEN1, MLH1, MSH2, MSH6, MUTYH, NBN, NF1, PALB2, PMS2,POLD1, POLE, PTEN, RAD51C, RAD51D, RET, SDHB, SDHC, SDHD, SMAD4, STK11,TP53, and VHL, wherein an adapter sequence is ligated to the ends of theplurality of amplicons; and (c) detecting at least one mutation in atleast one of the plurality of amplicons using high throughput massiveparallel sequencing. In some embodiments, the dried biological fluidsample is dried plasma, dried serum, or dried whole blood. In certainembodiments, the dried biological fluid sample is obtained from apatient having, or is suspected of having a hereditary cancer. Thehereditary cancer may be breast cancer, ovarian cancer, colon cancer, orskin cancer.

Additionally or alternatively, in some embodiments, the dried biologicalfluid sample on the absorbent tip of the microsampling device iscollected from a patient via fingerstick. In certain embodiments, themicrosampling device is a MITRA® tip. Elution of the dried biologicalfluid sample may be performed by contacting the absorbent tip of themicrosampling device with a lysis buffer and Proteinase K. In certainembodiments, the lysis buffer comprises guanidine hydrochloride,Tris·Cl, EDTA, Tween 20, and Triton X-100. In a further embodiment, thelysis buffer comprises 800 mM guanidine hydrochloride; 30 mM Tris·Cl, pH8.0; 30 mM EDTA, pH 8.0; 5% Tween 20; and 0.5% Triton X-100.

Additionally or alternatively, in some embodiments, elution of the driedbiological fluid sample is performed by contacting the absorbent tip ofthe microsampling device with the lysis buffer for up to 15 minutes at90° C. Additionally or alternatively, in certain embodiments, elution ofthe dried biological fluid sample is performed by contacting theabsorbent tip of the microsampling device with Proteinase K for up to 1hour at 56° C. In other embodiments, elution of the dried biologicalfluid sample is performed by contacting the absorbent tip of themicrosampling device with Proteinase K for up to 16-18 hours at 56° C.In some embodiments, the sample volume of the microsampling device is nomore than 10-20 μL.

In some embodiments of the method, no more than 400 ng of genomic DNA iseluted from the absorbent tip of the microsampling device. In otherembodiments of the method, about 100 ng to about 400 ng of genomic DNAis eluted from the absorbent tip of the microsampling device.

Additionally or alternatively, in some embodiments, the plurality ofamplicons further comprises a unique index sequence. In certainembodiments, the plurality of amplicons are enriched using a bait setcomprising nucleic acid sequences that are complementary to at least oneof the plurality of amplicons. In some embodiments, the nucleic acidsequences of the bait set are RNA baits, DNA baits, or a combinationthereof.

In one embodiment, the methods featured in the present technology areused in a multiplex, multi-gene assay format, e.g., assays thatincorporate multiple signals from a large number of diverse geneticalterations in a large number of genes.

In some embodiments of the method, the at least one mutation detected isa mutation in APC, ATM, BARD1, BMPR1A, BRCA1, BRCA2, BRIP1, CDH1, CDK4,CDKN2A (p14ARF and p16), CHEK2, EPCAM, MEN1, MLH1, MSH2, MSH6, MUTYH,NBN, NF1, PALB2, PMS2, POLD1, POLE, PTEN, RAD51C, RAD51D, RET, SDHB,SDHC, SDHD, SMAD4, STK11, TP53, and VHL.

In some embodiments, a single primer or one or both primers of a primerpair comprise a specific adapter sequence (also referred to as asequencing adapter) ligated to the 5′ end of the target specificsequence portion of the primer. This sequencing adapter is a shortoligonucleotide of known sequence that can provide a priming site forboth amplification and sequencing of the adjoining, unknown targetnucleic acid. As such, adapters allow binding of a fragment to a flowcell for next generation sequencing. Any adapter sequence may beincluded in a primer used in the present technology. In certainembodiments, amplicons corresponding to specific regions of APC, ATM,BARD1, BMPR1A, BRCA1, BRCA2, BRIP1, CDH1, CDK4, CDKN2A (p14ARF and p16),CHEK2, EPCAM, MEN1, MLH1, MSH2, MSH6, MUTYH, NBN, NF1, PALB2, PMS2,POLD1, POLE, PTEN, RAD51C, RAD51D, RET, SDHB, SDHC, SDHD, SMAD4, STK11,TP53, and VHL are amplified using primers that contain anoligonucleotide sequencing adapter to produce adapter tagged amplicons.

In other embodiments, the employed primers do not contain adaptersequences and the amplicons produced are subsequently (i.e. afteramplification) ligated to an oligonucleotide sequencing adapter on oneor both ends of the amplicons. In some embodiments, all forwardamplicons (i.e., amplicons extended from forward primers that hybridizedwith antisense strands of a target nucleic acid) contain the sameadapter sequence. In some embodiments when double stranded sequencing isperformed, all forward amplicons contain the same adapter sequence andall reverse amplicons (i.e., amplicons extended from reverse primersthat hybridized with sense strands of a target segment) contain anadapter sequence that is different from the adapter sequence of theforward amplicons. In some embodiments, the adapter sequences furthercomprise an index sequence (also referred to as an index tag, a“barcode” or a multiplex identifier (MID)).

In some embodiments, the adapter sequences are P5 and/or P7 adaptersequences that are recommended for Illumina sequencers (MiSeq andHiSeq). See, e.g., Williams-Carrier et al., Plant J., 63(1):167-77(2010). In some embodiments, the adapter sequences are P1, A, or IonXpress™ barcode adapter sequences that are recommended for LifeTechnologies sequencers. Other adapter sequences are known in the art.Some manufacturers recommend specific adapter sequences for use with theparticular sequencing technology and machinery that they offer.

Additionally or alternatively, in some embodiments of the above methods,amplicons corresponding to specific regions of APC, ATM, BARD1, BMPR1A,BRCA1, BRCA2, BRIP1, CDH1, CDK4, CDKN2A (p14ARF and p16), CHEK2, EPCAM,MEN1, MLH1, MSH2, MSH6, MUTYH, NBN, NF1, PALB2, PMS2, POLD1, POLE, PTEN,RAD51C, RAD51D, RET, SDHB, SDHC, SDHD, SMAD4, STK11, TP53, and VHL frommore than one sample are sequenced. In some embodiments, all samples aresequenced simultaneously in parallel.

In some embodiments of the above methods, amplicons corresponding tospecific regions of APC, ATM, BARD1, BMPR1A, BRCA1, BRCA2, BRIP1, CDH1,CDK4, CDKN2A (p14ARF and p16), CHEK2, EPCAM, MEN1, MLH1, MSH2, MSH6,MUTYH, NBN, NF1, PALB2, PMS2, POLD1, POLE, PTEN, RAD51C, RAD51D, RET,SDHB, SDHC, SDHD, SMAD4, STK11, TP53, and VHL from at least 1, 5, 10,20, 30 or up to 35, 40, 45, 48 or 50 different samples are amplified andsequenced using the methods described herein.

Additionally or alternatively, in some embodiments of the method,amplicons derived from a single sample may further comprise an identicalindex sequence that indicates the source from which the amplicon isgenerated, the index sequence for each sample being different from theindex sequences from all other samples. As such, the use of indexsequences permits multiple samples to be pooled per sequencing run andthe sample source subsequently ascertained based on the index sequence.In some embodiments, the Access Array™ System (Fluidigm Corp., SanFrancisco, Calif.) or the Apollo 324 System (Wafergen Biosystems,Fremont, Calif.) is used to generate a barcoded (indexed) ampliconlibrary by simultaneously amplifying the nucleic acids from the samplesin one set up.

In some embodiments, indexed amplicons are generated using primers (forexample, forward primers and/or reverse primers) containing the indexsequence. Such indexed primers may be included during librarypreparation as a “barcoding” tool to identify specific amplicons asoriginating from a particular sample source. When adapter-ligated and/orindexed primers are employed, the adapter sequence and/or index sequencegets incorporated into the amplicon (along with the target-specificprimer sequence) during amplification. Therefore, the resultingamplicons are sequencing-competent and do not require the traditionallibrary preparation protocol. Moreover, the presence of the index tagpermits the differentiation of sequences from multiple sample sources.

In some embodiments, the amplicons may be amplified withnon-adapter-ligated and/or non-indexed primers and a sequencing adapterand/or an index sequence may be subsequently ligated to one or both endsof each of the resulting amplicons. In some embodiments, the ampliconlibrary is generated using a multiplexed PCR approach.

Indexed amplicons from more than one sample source are quantifiedindividually and then pooled prior to high throughput sequencing. Assuch, the use of index sequences permits multiple samples (i.e., samplesfrom more than one sample source) to be pooled per sequencing run andthe sample source subsequently ascertained based on the index sequence.“Multiplexing” is the pooling of multiple adapter-tagged and indexedlibraries into a single sequencing run. When indexed primer sets areused, this capability can be exploited for comparative studies. In someembodiments, amplicon libraries from up to 48 separate sources arepooled prior to sequencing.

Following the production of an adapter tagged and, optionally indexed,amplicon library, the amplicons are sequenced using high throughput,massively parallel sequencing (i.e., next generation sequencing).Methods for performing high throughput, massively parallel sequencingare known in the art. In some embodiments of the method, the highthroughput massive parallel sequencing is performed using 454™ GS FLX™pyrosequencing, reversible dye-terminator sequencing, SOLiD sequencing,Ion semiconductor sequencing, Helioscope single molecule sequencing,sequencing by synthesis, sequencing by ligation, or SMRT™ sequencing. Insome embodiments, high throughput massively parallel sequencing may beperformed using a read depth approach.

Treatment for Hereditary Cancers

Disclosed herein are methods for determining whether a patient willbenefit from treatment with one or more anti-cancer therapeutic agents.

Examples of breast and ovarian cancer therapies are well known in theart and include surgery, radiation therapy, hormonal therapy,chemotherapy, immunotherapy or combinations thereof. Immunotherapeuticagents include antibodies, radioimmunoconjugates and immunocytokines.

Classes of chemotherapeutic agents can include alkylating agents,platinum agents, taxanes, vinca agents, anti-estrogen drugs, aromataseinhibitors, ovarian suppression agents, VEGF/VEGFR inhibitors, EGF/EGFRinhibitors, PARP inhibitors, cytostatic alkaloids, cytotoxicantibiotics, antimetabolites, endocrine/hormonal agents, bi sphosphonatetherapy agents and targeted biological therapy agents (e.g., therapeuticpeptides described in U.S. Pat. No. 6,306,832, WO 2012007137, WO2005000889, WO 2010096603 etc.).

Specific chemotherapeutic agents include, but are not limited to,cyclophosphamide, fluorouracil (or 5-fluorouracil or 5-FU),methotrexate, edatrexate (10-ethyl-10-deaza-aminopterin), thiotepa,carboplatin, cisplatin, taxanes, paclitaxel, protein-bound paclitaxel,docetaxel, vinorelbine, tamoxifen, raloxifene, toremifene, fulvestrant,gemcitabine, irinotecan, ixabepilone, temozolmide, topotecan,vincristine, vinblastine, eribulin, mutamycin, capecitabine,anastrozole, exemestane, letrozole, leuprolide, abarelix, buserlin,goserelin, megestrol acetate, risedronate, pamidronate, ibandronate,alendronate, denosumab, zoledronate, trastuzumab, tykerb, anthracyclines(e.g., daunorubicin and doxorubicin), bevacizumab, or combinationsthereof.

Combinational chemotherapeutic therapies can include AT: Adriamycin®(Doxorubicin) and Taxotere® (Docetaxel); AC: Adriamycin®, Cytoxan®(Cyclophosphamide); AC+Taxol®; AC+Taxotere®; CMF: Cytoxan®,Methotrexate, 5-fluorouracil; CEF: Cytoxan®, Ellence® (Epirubicin), andfluorouracil; EC: Ellence®, Cytoxan®; FAC: 5-fluorouracil, Adriamycin®,and Cytoxan®; GET: Gemzar® (Gemcitabine), Ellence®, and Taxol®; TC:Taxotere®, Cytoxan®; TC: Taxotere®, Paraplatin® (Carboplatin); TAC:Taxotere®, Adriamycin®, Cytoxan® or TCH: Taxotere®, Herceptin®(Trastuzumab), and Paraplatin®. Additional combination chemotherapeutictherapies for metastatic breast or ovarian cancer include: Taxol andXeloda® (Capecitabine); Taxotere and Xeloda®; Taxotere and Paraplatin®;Taxol® and Paraplatin®; Taxol® and Gemzar®; Abraxane® (Protein-boundPaclitaxel) and Xeloda®; Abraxane® and Paraplatin®; Camptosor®(Irinotecan) and Temodar® (Temozolomide); Gemzar® and Paraplatin® orIxempra® (Ixabepilone) and Xeloda®. In some embodiments, thechemotherapeutic agents include cyclophosphamide and 5-fluorouracil orinclude methotrexate, cyclophosphamide and 5-fluorouracil.

Non-limiting examples of anti-cancer drugs for treating skin cancerinclude aldesleukin, cobimetinib, dabrafenib, dacarbazine, fluorouracil,talimogene laherparepvec, imiquimod, recombinant Interferon Alfa-2b,ipilimumab, pembrolizumab, trametinib, nivolumab, peginterferon Alfa-2b,sonidegib, vismodegib, and vemurafenib.

Non-limiting examples of anti-cancer drugs for treating colon cancerinclude bevacizumab, capecitabine, cetuximab, irinotecan hydrochloride,leucovorin calcium, trifluridine and tipiracil hydrochloride,oxaliplatin, panitumumab, ramucirumab, regorafenib, and ziv-aflibercept.

In one aspect, the present disclosure provides a method for selecting apatient exhibiting cancer symptoms, or a patient at risk for hereditarycancer, for treatment with an anti-cancer therapeutic agent comprising(a) eluting a dried blood sample under conditions that result in therelease of genomic DNA from blood cells, wherein the dried blood sampleis collected from the patient with a volumetric absorptive microsamplingdevice; (b) isolating genomic DNA from the eluted dried blood sample;(c) generating a library comprising amplicons corresponding to each of aplurality of hereditary cancer-related genes comprising APC, ATM, BARD1,BMPR1A, BRCA1, BRCA2, BRIP1, CDH1, CDK4, CDKN2A (p14ARF and p16), CHEK2,EPCAM, MEN1, MLH1, MSH2, MSH6, MUTYH, NBN, NF1, PALB2, PMS2, POLD1,POLE, PTEN, RAD51C, RAD51D, RET, SDHB, SDHC, SDHD, SMAD4, STK11, TP53,and VHL, wherein an adapter sequence is ligated to the ends of theplurality of amplicons; (d) detecting at least one mutation in at leastone of the plurality of amplicons using high throughput massive parallelsequencing; and (e) selecting the patient for treatment with ananti-cancer therapeutic agent, if a mutation in at least one of theplurality of amplicons corresponding to one or more of APC, ATM, BARD1,BMPR1A, BRCA1, BRCA2, BRIP1, CDH1, CDK4, CDKN2A (p14ARF and p16), CHEK2,EPCAM MEN1, MLH1, MSH2, MSH6, MUTYH, NBN, NF1, PALB2, PMS2, POLD1, POLE,PTEN, RAD51C, RAD51D, RET, SDHB, SDHC, SDHD, SMAD4, STK11, TP53, and VHLis detected. In certain embodiments, the volumetric absorptivemicrosampling device is a MITRA® tip. In some embodiments, the patienthas, or is at risk for a hereditary cancer selected from the groupconsisting of breast cancer, ovarian cancer, skin cancer, or coloncancer.

Patients at risk for hereditary cancer include subjects having: (a) twoor more close relatives diagnosed with cancer; (b) multiple primarytumors; (c) bilateral or rare cancers; (d) familial incidences of cancerin multiple generations; (e) a constellation of tumors consistent with aspecific cancer syndrome; (f) certain ethnic backgrounds (e.g.,Ashkenazi Jewish ancestry); or cancers that manifest at a young age.

Cancer symptoms include, but are not limited to, persistent cough orblood-tinged saliva, change in bowel habits, bloody stool, anemia,breast lumps or breast discharge, testicular lumps, change in urinationfrequency, hematuria, hoarseness, persistent lumps or swollen glands,moles that bleed or have irregular edges, indigestion or difficultyswallowing, unusual vaginal bleeding or discharge, unexpected weightloss, night sweats, fever, persistent itching in the anal or genitalarea, non-healing sores, headaches, back pain, pelvic pain, andbloating.

In any of the above embodiments, the anti-cancer therapeutic agent isone or more agents selected from the group consisting ofcyclophosphamide, fluorouracil (or 5-fluorouracil or 5-FU),methotrexate, edatrexate (10-ethyl-10-deaza-aminopterin), thiotepa,carboplatin, cisplatin, taxanes, paclitaxel, protein-bound paclitaxel,docetaxel, vinorelbine, tamoxifen, raloxifene, toremifene, fulvestrant,gemcitabine, irinotecan, ixabepilone, temozolmide, topotecan,vincristine, vinblastine, eribulin, mutamycin, capecitabine,anastrozole, exemestane, letrozole, leuprolide, abarelix, buserlin,goserelin, megestrol acetate, risedronate, pamidronate, ibandronate,alendronate, denosumab, zoledronate, trastuzumab, tykerb,anthracyclines, bevacizumab, aldesleukin, cobimetinib, dabrafenib,dacarbazine, talimogene laherparepvec, imiquimod, recombinant InterferonAlfa-2b, ipilimumab, pembrolizumab, trametinib, nivolumab, peginterferonAlfa-2b, sonidegib, vismodegib, vemurafenib, cetuximab, irinotecanhydrochloride, leucovorin calcium, trifluridine and tipiracilhydrochloride, oxaliplatin, panitumumab, ramucirumab, regorafenib, andziv-aflibercept.

Kits

The present disclosure provides kits for detecting one or more mutationsin the plurality of hereditary cancer-related genes described herein, ina dried biological fluid sample. In some embodiments, the kits comprisea skin puncture tool, a volumetric absorptive microsampling device, alysis buffer, and proteinase K, wherein the plurality of hereditarycancer-related genes comprises APC, ATM, BARD1, BMPR1A, BRCA1, BRCA2,BRIP1, CDH1, CDK4, CDKN2A (p14ARF and p16), CHEK2, EPCAM MEN1, MLH1,MSH2, MSH6, MUTYH, NBN, NF1, PALB2, PMS2, POLD1, POLE, PTEN, RAD51C,RAD51D, RET, SDHB, SDHC, SDHD, SMAD4, STK11, TP53, and VHL. The lysisbuffer may comprise guanidine hydrochloride, Tris·Cl, EDTA, Tween 20,and Triton X-100. Alternatively, the lysis buffer may comprise 2.5-10%sodium dodecyl sulphate.

In some embodiments, the kits further comprise one or more componentsfor denaturing nucleoprotein complexes in cells present in the driedbiological fluid sample. Additionally or alternatively, in someembodiments, the kits further comprise one or more components forremoving protein contaminants, inactivating nuclease activity, and/orremoving biological and/or chemical contaminants present in the driedbiological fluid sample.

In some embodiments, the kits further comprise one or more primer pairsthat hybridize to one or more regions or exons of one or more of theplurality of hereditary cancer-related genes. Additionally oralternatively, in some embodiments, the kits further comprise one ormore bait sequences that hybridize to one or more regions or exons ofone or more of the plurality of hereditary cancer-related genes.

Particularly, in some embodiments, kits of the present technologycomprise one or more primer pairs or bait sequences that selectivelyhybridize to, and are useful in amplifying or capturing one or more ofthe genes selected from the group consisting of APC, ATM, BARD1, BMPR1A,BRCA1, BRCA2, BRIP1, CDH1, CDK4, CDKN2A (p14ARF and p16), CHEK2, EPCAMMEN1, MLH1, MSH2, MSH6, MUTYH, NBN, NF1, PALB2, PMS2, POLD1, POLE, PTEN,RAD51C, RAD51D, RET, SDHB, SDHC, SDHD, SMAD4, STK11, TP53, and VHL.

In some embodiments, the kits of the present technology comprise asingle primer pair or bait sequence that hybridizes to a region or exonof a single gene selected from the group consisting of APC, ATM, BARD1,BMPR1A, BRCA1, BRCA2, BRIP1, CDH1, CDK4, CDKN2A (p14ARF and p16), CHEK2,EPCAM, MEN1, MLH1, MSH2, MSH6, MUTYH, NBN, NF1, PALB2, PMS2, POLD1,POLE, PTEN, RAD51C, RAD51D, RET, SDHB, SDHC, SDHD, SMAD4, STK11, TP53,and VHL. In other embodiments, the kits of the present technologycomprise multiple primer pairs or bait sequences that hybridize to oneor more regions or exons of a single gene selected from the groupconsisting of APC, ATM, BARD1, BMPR1A, BRCA1, BRCA2, BRIP1, CDH1, CDK4,CDKN2A (p14ARF and p16), CHEK2, EPCAM, MEN1, MLH1, MSH2, MSH6, MUTYH,NBN, NF1, PALB2, PMS2, POLD1, POLE, PTEN, RAD51C, RAD51D, RET, SDHB,SDHC, SDHD, SMAD4, STK11, TP53, and VHL. In certain embodiments, thekits of the present technology comprise multiple primer pairs or baitsequences comprising a single primer pair or bait sequence thatspecifically hybridizes to a region or exon of a single gene for each ofAPC, ATM, BARD1, BMPR1A, BRCA1, BRCA2, BRIP1, CDH1, CDK4, CDKN2A (p14ARFand p16), CHEK2, EPCAM, MEN1, MLH1, MSH2, MSH6, MUTYH, NBN, NF1, PALB2,PMS2, POLD1, POLE, PTEN, RAD51C, RAD51D, RET, SDHB, SDHC, SDHD, SMAD4,STK11, TP53, and VHL. In certain embodiments, the kits of the presenttechnology comprise multiple primer pairs or bait sequences comprisingmore than one primer pair or more than one bait sequence that hybridizesto one or more regions or exons for each of APC, ATM, BARD1, BMPR1A,BRCA1, BRCA2, BRIP1, CDH1, CDK4, CDKN2A (p14ARF and p16), CHEK2, EPCAM,MEN1, MLH1, MSH2, MSH6, MUTYH, NBN, NF1, PALB2, PMS2, POLD1, POLE, PTEN,RAD51C, RAD51D, RET, SDHB, SDHC, SDHD, SMAD4, STK11, TP53, and VHL.

Thus, it is contemplated herein that the kits of the present technologycan comprise primer pairs or bait sequences that recognize andspecifically hybridize to one or more regions or exons of one or moregenes selected from the group consisting APC, ATM, BARD1, BMPR1A, BRCA1,BRCA2, BRIP1, CDH1, CDK4, CDKN2A (p14ARF and p16), CHEK2, EPCAM, MEN1,MLH1, MSH2, MSH6, MUTYH, NBN, NF1, PALB2, PMS2, POLD1, POLE, PTEN,RAD51C, RAD51D, RET, SDHB, SDHC, SDHD, SMAD4, STK11, TP53, and VHL.

In any of the above embodiments of the kits of the present technology,the volumetric absorptive microsampling device is a MITRA® tip.

In some embodiments, the kits may comprise a plurality of volumetricabsorptive microsampling devices, each having a hollow holder at theproximal end and an absorbent tip at the distal end. The absorbent tipcomprises a hydrophilic, polymeric material configured to absorb 30microliters or less of blood within about 10 seconds or less. The kitalso includes a container having a plurality of compartments. Eachcompartment is configured to releasably engage a volumetric absorptivemicrosampling device. The container is configured to prevent theabsorbent tips of the microsampling devices from abutting thecompartment within which the microsampling device is placed.

Additionally or alternatively, in certain embodiments, the kits mayinclude a plurality of access ports with each port associated with anindividual compartment. Each port is located to allow printing onto theholder of a volumetric absorptive microsampling device present withinthe compartment with which the port is associated. In certainembodiments, the holder of a volumetric absorptive microsampling devicehas a plurality of ribs extending along a length of the holder with theribs configured to keep the absorbent tip from contacting walls of thecontainer. The container preferably has two parts configured to formtubular shaped compartments. The container may have a first part with aplurality of elongated mounting protrusions each extending along aportion of a different compartment. The hollow end of the holder of thevolumetric absorptive microsampling device fits onto the mountingprotrusion to releasably fasten the holder onto the mounting protrusion.

In some embodiments, the kit comprises liquid medium containing the atleast one target-specific nucleic acid probe in a concentration of 250nM or less. With such a kit, the probes are provided in the requiredamount to perform reliable multiplex detection reactions according tothe present technology.

In some embodiments, the kits further comprise buffers, enzymes havingpolymerase activity, enzymes having polymerase activity and lacking5′→3′ exonuclease activity or both 5′→3′ and 3′→5′ exonuclease activity,enzyme cofactors such as magnesium or manganese, salts, chain extensionnucleotides such as deoxynucleoside triphosphates (dNTPs), modifieddNTPs, nuclease-resistant dNTPs or labeled dNTPs, necessary to carry outan assay or reaction, such as amplification and/or detection of one ormore mutations in the plurality of hereditary cancer-related genesdescribed herein, in a dried biological fluid sample.

In one embodiment, the kits of the present technology further comprise apositive control nucleic acid sequence and a negative control nucleicacid sequence to ensure the integrity of the assay during experimentalruns. The kit may also comprise instructions for use, software forautomated analysis, containers, packages such as packaging intended forcommercial sale and the like.

The kit may further comprise one or more of: wash buffers and/orreagents, hybridization buffers and/or reagents, labeling buffers and/orreagents, and detection means. The buffers and/or reagents are usuallyoptimized for the particular amplification/detection technique for whichthe kit is intended. Protocols for using these buffers and reagents forperforming different steps of the procedure may also be included in thekit.

The kits of the present technology may include components that are usedto prepare nucleic acids from a dried biological fluid sample for thesubsequent amplification and/or detection of alterations in targetnucleic acid sequences corresponding to the plurality of hereditarycancer-related genes disclosed herein. Such sample preparationcomponents can be used to produce nucleic acid extracts from driedbiological fluid samples, such as dried serum, dried plasma, or driedwhole blood. The test samples used in the above-described methods willvary based on factors such as the assay format, nature of the detectionmethod, and the specific cells or extracts used as the test sample to beassayed. Methods of extracting nucleic acids from samples are well knownin the art and can be readily adapted to obtain a sample that iscompatible with the system utilized. Automated sample preparationsystems for extracting nucleic acids from a test sample are commerciallyavailable, e.g., Roche Molecular Systems' COBAS AmpliPrep System,Qiagen's BioRobot 9600, Qiagen's BioRobot EZ1, QIAsymphony®, and AppliedBiosystems' PRISM™ 6700 sample preparation system.

EXAMPLES Example 1 Extraction of Genomic DNA from Dried Blood SamplesCollected Using MITRA® Tips

This Example demonstrates that the methods of the present technology areuseful for extracting high yields of genomic DNA from a dried biologicalfluid sample (e.g., dried blood) collected using a volumetric absorptivemicrosampling device.

A total of four human subjects were enrolled in the study. Three MITRA®tips of blood were collected from each of 4 blood donors in order tosimultaneously test three extraction methods. A fixed volume of 10 μL ofblood was collected on each MITRA® Tip collection device viafingerstick. After drying the blood samples, the absorbent tips of theMITRA® Tip collection devices were then placed in 180 μL Buffer G2 (alysis buffer containing 800 mM guanidine hydrochloride; 30 mM Tris·Cl,pH 8.0; 30 mM EDTA, pH 8.0; 5% Tween 20; 0.5% Triton X-100) and werevortexed for 15 seconds. The remaining sample processing steps for eachof the three extraction methods are summarized below:

Step Method 1 Method 2 Method 3 1 Incubate MITRA ® Tip in — IncubateMITRA ® Tip in Buffer G2 at 90° C. for 15 min Buffer G2 at 90° C. for 15min 2 Vortex for 15 sec — Vortex for 15 sec 3 Add 10 μL Proteinase K 4Vortex for 15 sec 5 Incubate with Proteinase Incubate with ProteinaseIncubate with Proteinase K at 56° C. for 1 hour K at 56° C. for 1 hour Kat 56° C. Overnight 6 Vortex 15 sec 7 Aliquot cell lysate to new tube 8Perform remaining genomic DNA extraction on EZ1 ® Biorobot using TissueDNA protocol

Extracted genomic DNA was then quantified using Qubit® dsDNA HS AssayKit, which uses a dsDNA intercalating dye that only fluoresces in thepresence of dsDNA. Therefore, quantitation of dsDNA using the Qubit®dsDNA HS Assay Kit is not affected by RNA, proteins, salts, or othercontaminants that may affect other quantitation methods. Table 1 andFIG. 1 demonstrate that the DNA yield obtained from each MITRA® tipvaried according to the extraction method. It was determined thatextraction method 3 (Incubation of MITRA® Tip with Buffer G2 at 90° C.for 15 min, and with Proteinase K at 56° C. overnight) yielded thehighest quantity of DNA.

TABLE 1 Range of DNA yield obtained per MITRA ® Tip Extraction MethodTotal DNA yield (ng) 1 111-248 2 163-210 3 222-390

Additionally, FIG. 3 demonstrates that DNA yields recovered from MITRA®Tips would vary significantly depending on the lysis buffer, lysisperiod, extraction platform, and the number of MITRA® Tips utilizedduring extraction procedure. For example, DNA yields recovered from asingle MITRA® Tip incubated with Qiagen ATL buffer (comprising 2.5-10%sodium dodecyl sulphate) for 1 hour was higher in the Qiagen platform(between 110-456 ng) compared to the Roche MagNA Pure platform (<30 ng).In contrast, DNA yields obtained with certain lysis buffers, such asRoche Lysis Buffer, Roche External Lysis Buffer, and Reliaprep™(Promega), were low (<10 ng). See FIG. 3.

These results demonstrate that the methods of the present technology areuseful for extracting high yields of genomic DNA from a dried biologicalfluid sample (e.g., dried blood) collected using a volumetric absorptivemicrosampling device.

Example 2 Detection of Hereditary Cancer-Related Mutations using DriedBlood Samples Extracted from MITRA® Tips

Genomic DNA was extracted from dried blood samples collected from eachdonor via MITRA® tips using extraction method 3 (incubation of MITRA®Tip with Buffer G2 at 90° C. for 15 min, and with Proteinase K at 56° C.overnight) as described in Example 1. DNA extracted from 2 MITRA® tipsper donor was pooled. Pooled DNA was then tested on the MyVantage™Hereditary Comprehensive Cancer Panel, which assesses 731 ampliconscorresponding to a plurality of 34 hereditary cancer-related genescomprising APC, ATM, BARD1, BMPR1A, BRCA1, BRCA2, BRIP1, CDH1, CDK4,CDKN2A (p14ARF and p16), CHEK2, EPCAM MEN1, MLH1, MSH2, MSH6, MUTYH,NBN, NF1, PALB2, PMS2, POLD1, POLE, PTEN, RAD51C, RAD51D, RET, SDHB,SDHC, SDHD, SMAD4, STK11, TP53, and VHL. Next generation sequencing wasperformed on the Illumina NextSeq® Sequencer. Quality metrics forassessing successful sequencing of a covered region included a minimumof 20 reads per covered position in a given region.

Results. FIG. 2(a) shows that 100% of the 731 assessed regions hadpassed the QC criteria in three samples. One sample had 730 out of 731assessed regions pass the QC criteria. The sequencing performance ofsamples subjected to single tip versus dual tip extraction was alsocompared. FIG. 2(b) and FIG. 4(b) show that 100% of the 731 assessedregions had passed the QC criteria, regardless of whether single tipextraction or dual tip extraction was employed.

FIG. 5 demonstrates that 10 kb and 18 kb amplicons corresponding toCHEK2 and PMS2 target regions respectively, were effectively amplifiedvia long-range PCR when using dried blood samples eluted from MITRA®tips. FIG. 6 demonstrates that genomic DNA extracted from dried bloodsamples eluted from MITRA® tips showed sufficient coverage of severalCHEK2 and PMS2 exons.

Further, FIG. 7 demonstrates that the minimum read coverage per targetregion using DNA obtained from MITRA® tips wicked with EDTA whole bloodwas comparable to that observed with DNA obtained from blood collectedby MITRA® tips via fingerstick. Thus, these results demonstrate that themethods of the present technology are effective in detecting hereditarycancer-related mutations in dried biological fluid samples containingknown PCR-inhibitors such as EDTA.

These results demonstrate that the methods of the present technology arecapable of detecting at least one mutation in the plurality ofhereditary cancer-related genes described herein, in a small-volumedried biological fluid sample that is collected with a volumetricabsorptive microsampling device (e.g., MITRA® Tip).

Example 3 Detection of Hereditary Cancer-Related Mutations using DriedBlood Samples Extracted from MITRA® Tips via QIAsymphony® Platform

Genomic DNA was extracted from 20 μL dried blood samples collected viaMITRA® tips using the QIAsymphony® platform (which permits automation of96 samples at a time). MITRA® tips were incubated with Qiagen ATL bufferovernight at 56° C. DNA extraction was subsequently performed on theQIAsymphony® platform according to manufacturer's instructions. Allsamples were subjected to single tip extraction, and resulted in DNAyields ranging between 109-358 ng. DNA was then tested on the MyVantage™Hereditary Comprehensive Cancer Panel and next generation sequencing wasperformed on the Illumina NextSeq® Sequencer. Quality metrics forassessing successful sequencing of a covered region included a minimumof 20 reads per covered position in a given region.

Results. FIG. 4(a) demonstrates that DNA input levels as low as 109 ng(obtained from a single MITRA® tip) displayed sufficient coverage on theMyVantage™ Hereditary Comprehensive Cancer Panel for both singlenucleotide variation (SNV) and insertion/deletion (INDEL) detection.

These results demonstrate that the methods of the present technology arecapable of detecting at least one mutation in the plurality ofhereditary cancer-related genes described herein, in a small-volumedried biological fluid sample that is collected with a volumetricabsorptive microsampling device (e.g., MITRA® Tip).

EQUIVALENTS

The present technology is not to be limited in terms of the particularembodiments described in this application, which are intended as singleillustrations of individual aspects of the present technology. Manymodifications and variations of this present technology can be madewithout departing from its spirit and scope, as will be apparent tothose skilled in the art. Functionally equivalent methods andapparatuses within the scope of the present technology, in addition tothose enumerated herein, will be apparent to those skilled in the artfrom the foregoing descriptions. Such modifications and variations areintended to fall within the scope of the present technology. It is to beunderstood that this present technology is not limited to particularmethods, reagents, compounds compositions or biological systems, whichcan, of course, vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular embodimentsonly, and is not intended to be limiting.

The terms “comprising,” “including,” “containing,” etc. shall be readexpansively and without limitation. Additionally, the terms andexpressions employed herein have been used as terms of description andnot of limitation, and there is no intention in the use of such termsand expressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the disclosure claimed.

In addition, where features or aspects of the disclosure are describedin terms of Markush groups, those skilled in the art will recognize thatthe disclosure is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

As will be understood by one skilled in the art, for any and allpurposes, particularly in terms of providing a written description, allranges disclosed herein also encompass any and all possible subrangesand combinations of subranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” “greater than,” “less than,” and the like,include the number recited and refer to ranges which can be subsequentlybroken down into subranges as discussed above. Finally, as will beunderstood by one skilled in the art, a range includes each individualmember. Thus, for example, a group having 1-3 cells refers to groupshaving 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers togroups having 1, 2, 3, 4, or 5 cells, and so forth.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

1. A method for detecting at least one mutation in a plurality ofhereditary cancer-related genes in a dried biological fluid samplecomprising (a) extracting genomic DNA from a dried biological fluidsample eluted from an absorbent tip of a microsampling device; (b)generating a library comprising amplicons corresponding to each of theplurality of hereditary cancer-related genes, said plurality ofhereditary cancer-related genes comprising APC, ATM, BARD1, BMPR1A,BRCA1, BRCA2, BRIP1, CDH1, CDK4, CDKN2A (p14ARF and p16), CHEK2, EPCAM,MEN1, MLH1, MSH2, MSH6, MUTYH, NBN, NF1, PALB2, PMS2, POLD1, POLE, PTEN,RAD51C, RAD51D, RET, SDHB, SDHC, SDHD, SMAD4, STK11, TP53, and VHL,wherein an adapter sequence is ligated to the ends of the plurality ofamplicons; and (c) detecting at least one mutation in at least one ofthe plurality of amplicons using high throughput massive parallelsequencing.
 2. The method of claim 1, wherein the dried biological fluidsample is obtained from a patient having, or is suspected of having ahereditary cancer.
 3. The method of claim 1 or 2, wherein the driedbiological fluid sample is dried plasma, dried serum, or dried wholeblood.
 4. The method of any one of claims 1-3, wherein the driedbiological fluid sample on the absorbent tip of the microsampling deviceis collected from a patient via fingerstick.
 5. The method of any one ofclaims 1-4, wherein elution of the dried biological fluid sample isperformed by contacting the absorbent tip of the microsampling devicewith a lysis buffer and Proteinase K.
 6. The method of claim 5, whereinthe lysis buffer comprises guanidine hydrochloride, Tris·Cl, EDTA, Tween20, and Triton X-100.
 7. The method of claim 5 or 6, wherein elution ofthe dried biological fluid sample is performed by contacting theabsorbent tip of the microsampling device with the lysis buffer for upto 15 minutes at 90° C.
 8. The method of any one of claims 5-7, whereinelution of the dried biological fluid sample is performed by contactingthe absorbent tip of the microsampling device with Proteinase K for upto 1 hour at 56° C.
 9. The method of any one of claims 5-7, whereinelution of the dried biological fluid sample is performed by contactingthe absorbent tip of the microsampling device with Proteinase K for upto 16-18 hours at 56° C.
 10. The method of any one of claims 1-9,wherein the microsampling device is a MITRA® tip.
 11. The method of anyone of claims 1-10, wherein the sample volume of the microsamplingdevice is no more than 10-20 μL.
 12. The method of any one of claims2-11, wherein the hereditary cancer is breast cancer, ovarian cancer,colon cancer, or skin cancer.
 13. The method of any one of claims 1-12,wherein no more than 400 ng of genomic DNA is eluted from the absorbenttip of the microsampling device.
 14. The method of any one of claims1-12, wherein about 100 ng to about 400 ng of genomic DNA is eluted fromthe absorbent tip of the microsampling device.
 15. The method of any oneof claims 1-14, wherein the high throughput massive parallel sequencingis performed using pyrosequencing, reversible dye-terminator sequencing,SOLiD sequencing, Ion semiconductor sequencing, Helioscope singlemolecule sequencing, sequencing by synthesis, sequencing by ligation, orSMUT″' sequencing.
 16. The method of any one of claims 1-15, wherein theadapter sequence is a P5 adapter, P7 adapter, P1 adapter, A adapter, orIon Xpress™ barcode adapter.
 17. The method of any one of claims 1-16,wherein the plurality of amplicons further comprise a unique indexsequence.
 18. The method of any one of claims 1-17, wherein theplurality of amplicons are enriched using a bait set comprising nucleicacid sequences that are complementary to at least one of the pluralityof amplicons.
 19. The method of claim 18, wherein the nucleic acidsequences of the bait set are RNA baits, DNA baits, or a combinationthereof.
 20. A method for detecting at least one mutation in a pluralityof hereditary cancer-related genes in a dried biological fluid samplecomprising isolating genomic DNA from a dried biological fluid sampleeluted from an absorbent tip of a microsampling device with a lysisbuffer and Proteinase K, wherein the plurality of hereditarycancer-related genes comprises APC, ATM, BARD1, BMPR1A, BRCA1, BRCA2,BRIP1, CDH1, CDK4, CDKN2A (p14ARF and p16), CHEK2, EPCAM, MEN1, MLH1,MSH2, MSH6, MUTYH, NBN, NF1, PALB2, PMS2, POLD1, POLE, PTEN, RAD51C,RAD51D, RET, SDHB, SDHC, SDHD, SMAD4, STK11, TP53, and VHL.
 21. Themethod of claim 20, wherein the at least one mutation in the pluralityof hereditary cancer-related genes is detected using high throughputmassive parallel sequencing.
 22. The method of claim 20 or 21, whereinthe lysis buffer comprises guanidine hydrochloride, Tris·Cl, EDTA, Tween20, and Triton X-100.
 23. A method for selecting a patient exhibitingcancer symptoms, or a patient at risk for hereditary cancer, fortreatment with an anti-cancer therapeutic agent comprising (a) eluting adried blood sample under conditions that result in the release ofgenomic DNA from blood cells, wherein the dried blood sample iscollected from the patient with a volumetric absorptive microsamplingdevice; (b) isolating genomic DNA from the eluted dried blood sample;(c) generating a library comprising amplicons corresponding to each of aplurality of hereditary cancer-related genes comprising APC, ATM, BARD1,BMPR1A, BRCA1, BRCA2, BRIP1, CDH1, CDK4, CDKN2A (p14ARF and p16), CHEK2,EPCAM, MEN1, MLH1, MSH2, MSH6, MUTYH, NBN, NF1, PALB2, PMS2, POLD1,POLE, PTEN, RAD51C, RAD51D, RET, SDHB, SDHC, SDHD, SMAD4, STK11, TP53,and VHL, wherein an adapter sequence is ligated to the ends of theplurality of amplicons; (d) detecting at least one mutation in at leastone of the plurality of amplicons using high throughput massive parallelsequencing; and (e) selecting the patient for treatment with ananti-cancer therapeutic agent, if a mutation in at least one of theplurality of amplicons corresponding to one or more of APC, ATM, BARD1,BMPR1A, BRCA1, BRCA2, BRIP1, CDH1, CDK4, CDKN2A (p14ARF and p16), CHEK2,EPCAM, MEN1, MLH1, MSH2, MSH6, MUTYH, NBN, NF1, PALB2, PMS2, POLD1,POLE, PTEN, RAD51C, RAD51D, RET, SDHB, SDHC, SDHD, SMAD4, STK11, TP53,and VHL is detected.
 24. The method of claim 23, wherein the volumetricabsorptive microsampling device is a MITRA® tip.
 25. The method of anyone of claims 23-24, wherein the patient has, or is at risk for ahereditary cancer selected from the group consisting of breast cancer,ovarian cancer, skin cancer, or colon cancer.
 26. The method of any oneof claims 23-25, wherein the anti-cancer therapeutic agent is one ormore agents selected from the group consisting of cyclophosphamide,fluorouracil (or 5-fluorouracil or 5-FU), methotrexate, edatrexate(10-ethyl-10-deaza-aminopterin), thiotepa, carboplatin, cisplatin,taxanes, paclitaxel, protein-bound paclitaxel, docetaxel, vinorelbine,tamoxifen, raloxifene, toremifene, fulvestrant, gemcitabine, irinotecan,ixabepilone, temozolmide, topotecan, vincristine, vinblastine, eribulin,mutamycin, capecitabine, anastrozole, exemestane, letrozole, leuprolide,abarelix, buserlin, goserelin, megestrol acetate, risedronate,pamidronate, ibandronate, alendronate, denosumab, zoledronate,trastuzumab, tykerb, anthracyclines, bevacizumab, aldesleukin,cobimetinib, dabrafenib, dacarbazine, talimogene laherparepvec,imiquimod, recombinant Interferon Alfa-2b, ipilimumab, pembrolizumab,trametinib, nivolumab, peginterferon Alfa-2b, sonidegib, vismodegib,vemurafenib, cetuximab, irinotecan hydrochloride, leucovorin calcium,trifluridine and tipiracil hydrochloride, oxaliplatin, panitumumab,ramucirumab, regorafenib, and ziv-aflibercept.
 27. A kit for detectingat least one mutation in a plurality of hereditary cancer-related genesin a dried biological fluid sample comprising a skin puncture tool, avolumetric absorptive microsampling device, a lysis buffer, andproteinase K, wherein the plurality of hereditary cancer-related genescomprises APC, ATM, BARD1, BMPR1A, BRCA1, BRCA2, BRIP1, CDH1, CDK4,CDKN2A (p14ARF and p16), CHEK2, EPCAM, MEN1, MLH1, MSH2, MSH6, MUTYH,NBN, NF1, PALB2, PMS2, POLD1, POLE, PTEN, RAD51C, RAD51D, RET, SDHB,SDHC, SDHD, SMAD4, STK11, TP53, and VHL.
 28. The kit of claim 27,further comprising one or more primer pairs that hybridize to one ormore regions or exons of one or more of the plurality of hereditarycancer-related genes.
 29. The kit of claim 27 or 28, further comprisingone or more bait sequences that hybridize to one or more regions orexons of one or more of the plurality of hereditary cancer-relatedgenes.
 30. The kit of any one of claims 27-29, wherein the volumetricabsorptive microsampling device is a MITRA® tip.
 31. The kit of any oneof claims 27-30, wherein the lysis buffer comprises guanidinehydrochloride, Tris·Cl, EDTA, Tween 20, and Triton X-100.